The Role of CIP4 in Osteosarcoma Metastases

By Nadezhda V. Koshkina, PhD

Introduction

Osteosarcoma (OS) is the most frequent bone tumor that predominantly targets the adolescent age group. The lung is the most common and often the only site of metastasis for OS. This type of metastasis continues to confer a generally poor prognosis despite decades of trials using intensified dosing, different timing and variations in combinations of chemical agents. The response rate for patients who present with localized disease remains unchanged at approximately 65% for more than 20 years. The prognosis for OS patients who present with metastases is significantly worse, with few survivors. This indicates the urgency of developing novel treatment approaches for these patients.

Lung metastases in osteosarcoma patients indicate poor prognosis. These patients represent a real challenge for physicians, because metastatic disease is frequently resistant to chemotherapy. In the last 20 years, modifications to the standard treatments for these patients have not made any significant changes in prognosis. It is our goal to understand the mechanisms of metastases and, based on this knowledge, identify new targets that can be used for the development of new therapeutic approaches to treat metastatic osteosarcoma.

A recent breakthrough was achieved by adding liposomal muramyltripeptide (MTP) to the standard chemotherapy for patients with OS.1 The use of this drug was recently approved by the European Medicines Agency. The idea of using this agent in OS came from the observation that many tumors, including OS, are immunologically "quiet", i.e. there is negligible presence of inflammatory cells in the tumor mass. Therefore, introduction of immonomodulators like MTP to patients with OS might activate immune cells and increase their infiltration into tumors. In preclinical and clinical studies it was proven that MTP was able to activate monocytes and macrophages to become tumoricidal for OS.2-4 This is a bright example of how understanding the biology of tumors can contribute to the development of novel therapies.

We use similar approach in our studies. We recently found that one of the cytoskeleton molecules, Cdc-42 interacting protein 4 (CIP4), is expressed in different OS tumor cells and showed a trend for a higher expression pattern in metastatic sublines (unpublished data). The Liddy Shriver Sarcoma Initiative, together with the FOSTER Foundation, supported our proposal to study the role of CIP4 in OS tumorigenesis and metastogenesis.

Figure 1: Inhibition of CIP4 reduced the growth of osteosarcoma...

Despite the fact that CIP4 was discovered in 1997,5 most of the studies on its functional activity were done in macrophages, and very little is currently known about its role in tumor cells. It was shown that CIP4 is required to coordinate membrane tubulation with reorganization of the actin cytoskeleton during endocytosis. It binds to cell membrane lipids and promotes membrane invagination and formation of tubules (for details see review article Ref. 6). In order to understand the function of CIP4 in OS, we first transfected highly aggressive mouse OS cells, DLM8 with the plasmid that downregulated expression of CIP4 protein (DLM8/shCIP4). The behavior of these cells was initially studied in tissue culture. We found that the growth rate of DLM8/shCIP4 cells was reduced by 30% when compared with non-transfected DLM8 cells or DLM8 cells transfected with control plasmid (DLM8/sh). When DLM8/shCIP4 cells were injected into mice subcutaneously they formed tumors that grew significantly slower than DLM8 or DLM8/sh control cells (Fig. 1 , p<0.02). This is a new finding, which needs further investigation of the mechanism. We also found that inhibition of CIP4 expression in DLM8 cells decreased their mobility and invasion properties in vitro, which indicates on their ability to impair metastases growth.

Figure 2: Downregulation of CIP4 expression in osteosarcoma...

Physiological function of CIP4 is associated with actin. In fact, it promotes Cdc42-induced actin polymerization.6 Actin polymerization is required for the formation of podosomes, actin-rich adhesion structures specific to monocyte-derived cells.7 Podosomes are necessary for directional movement of the cells, such as macrophages, and in osteoclasts they are thought to aid in the creation of sealing rings associated with the area of bone resorption8 In tumor cells podosome-similar structures are called invadopodia or "invasive feet.9" When we compared the actin filaments structures in DLM8/shCIP4 cells with wild-type DLM8 or DLM8/si control cells we observed lower perinuclear actin stress filaments formation and lower actin bridge to the lipid bilayer of the lipid cell membrane in them (Fig. 2). We also observed that downregulation of CIP4 changed cell membrane morphology in DLM8 cells – they had less protrusions and smoother cell surface than control cells. All these findings indicate that CIP4 may also play a role in metastatic behavior of these cells. We are currently working on studying the effect of CIP4 on the growth of OS metastases cells in vivo.

Summary and Conclusions

OS remains a devastating disease, and better understanding of its biology is required for the development of novel therapeutic approaches. The information obtained in this study indicates that CIP4 may become a new target for OS treatment. We obtained the evidence that inhibition of CIP4 changes cytoskeleton arrangement of OS cells and alters the behavior of these cells in tissue culture changing its potential to impair growth of OS metastases. CIP4 caused the reduction of the primary tumor growth in vitro and in xenograft subcutaneous animal model. Work should be done in the future to better understand the mechanisms of CIP4 activity in OS tumors and to characterize the effect of CIP4 on the growth of OS metastases.

CIP4 may become a novel target for osteosarcoma treatment. Due to its effect on the cytoskeletal structure, it changes the proliferation growth of OS in vitro and in animal models. Our preliminary findings indicate its potential to affect metastatic osteosarcoma growth in the lungs. Currently the role and mechanisms of cytoskeletal remodeling during the metastatic process are receiving much attention, but such information specific to sarcomas is limited, and further study is needed.

Novel Targets to Treat Osteosarcoma Lung Metastases

By Nadezhda V. Koshkina, PhD and Seth Corey, MD

Introduction

Osteosarcoma (OS) is one of the most common types of bone cancer. Currently, the standard therapy for OS patients includes surgery with pre- and post-surgery chemotherapy consisting of a combination of adriamycin, cisplatin, ifosfamide, and methotrexate. Although the combination of these drugs has significantly improved the 5-year disease-free survival rate up to 65% during the last 30 years, patients with metastatic disease fare very poorly. The most common site for OS metastases is the lung. The best case scenario for patients with metastases is a survival rate of 30% assuming complete resection of lung metastases. OS lung metastases are usually resistant to the treatment. Trials of new therapeutic agents that showed promising results in other types of tumors have had little effect on patients with OS metastases. Therefore, investigation into the biology of OS as well as in the identification of molecular features that correlate with disease prognosis will likely yield important findings that will impact therapy in the future.

Prognostic molecular markers for osteosarcoma. Studies of molecular mechanisms relating to the progression of OS during the last decade have identified several important factors that provided important information to assist the development of novel agents which either block or enhance those clinically relevant pathways. Among them are matrix metalloproteinases (MMPs), the enzymes involved in the break down of the compounds between cells. These enzymes play an important role during normal tissue remodeling. However, increased production of MMPs has been recognized as an important factor in cancer invasion and metastases. Elevated levels of one of them, MMP-9 correlated with poor prognosis in OS patients and were associated with increased risk of metastasis formation.1,2Stromal-derived factor-1 (SDF-1), a chemokine which is abundantly produced in the lungs, was shown to work as a chemoattractant for OS tumor cells leading them to migrate to the lungs and form metastases in this organ.3 In other studies it was shown that elevated levels of SDF-1 may result in the induction of MMPs production by tumor cells thus helping tumor cells to extravasate from the primary site into the blood stream and invade into the lungs.4 Activation of SDF-1 synthesis in OS tumor samples correlated with reduced overall survival and with the increased incidence of metastases in OS patients.5 The value of this information lies in both the prognostic potential, and the therapeutic relevance, because MMP-9 can be inhibited by histone deacetylase inhibitors and sulfoglycosamine,6 new anticancer agents which are currently undergoing clinical trials. Several types of SDF-1 peptide antagonists were recently developed and showed promising preclinical results in OS lung metastases in animal models.7

P-glycoprotein (P-gp) is another example of the factor that was identified as an indicator of the clinical outcome for OS patients. P-pg protein is responsible for drug efflux from cancer cells. Analysis of P-gp expression in OS tumors from patients before and after treatment and their overall response to the therapy allowed investigators to conclude that P-gp may be responsible for the development of drug resistance in OS tumor cells and suggest that clinical testing for P-gp may, in future, influence the choice and dose of chemotherapeutic agents for OS patients.8 Ferrari et al. noticed higher levels of P-gp levels in lung metastases than in the primary tumors of OS patients, which could explain poor response of patients with lung metastases to the salvage chemotherapy.9

Along with molecular prognostic factors for OS mentioned here, numerous others have been described (Table 1). Despite a long list of these factors none of them seems to be dominant and it looks that in future most likely combination of these factors should be considered for prognosis and selection of therapeutic options for individual OS cases.

Fas Signaling as a Novel Target in Osteosarcoma

In our recent studies we have identified that the Fas death receptor, which triggers cell death after binding with Fas ligand (FasL), plays an important role in OS metastasis formation. Using mouse models of OS lung metastases, we demonstrated that only Fas-negative tumor cells were able to survive and form lung metastases; Fas-positive tumor cells died in the lungs because of the interaction of their Fas receptor with FasL.30 It is important to note that FasL expression is limited to very few organs in the human body, including the lungs. Manipulations with Fas expression in tumor cells substantially changed their metastatic behavior in mice. For, example upregulation of Fas expression in metastatic OS cells significantly inhibited their metastatic potential (Fig. 1A).33 In contrast, inhibition of the Fas signaling in non-metastatic OS cells increased their metastatic behavior (Fig. 1B).33

Figures 1A and 1B: Alterations in the Fas Signaling...

Discussion of Figures 1a and 1b: Alterations in the Fas Signaling changes metastatic behavior of osteosarcoma cells in vivo. (A) LM7 human osteosarcoma cells were stably transfected with control neo-plasmid (LLM7-neo) or with Fas-plasmid (LM7-Fas). Cells were injected intravenously into immunodeficient mice and after 10 weeks their lungs were examined for metastases. All mice injected with LM7 or LM7-neo cells developed numerous visible metastases in the lungs. In contrast, fewer mice injected with LM7-Fas cells developed visible lung metastases and the number and size of these metastases was significantly smaller then in LM7 and LM7-neo groups. (B) K7 mouse osteosarcoma cells were transfected with control neo-plasmid or with FADD-dominant-negative plasmid (FDN), which inhibited Fas signaling. Stably transfected control clones K7/neo1 and K7/neo5 and two clones K7/FDN1 and K7/FDN5 were selected and then injected intravenously into immunocompetent mice. After 4 weeks animal lungs were examined for metastases. K7 and K7/neo groups of mice had less than 10% incidence of visible lung metastases, whereas all mice in K7/FDN groups developed visible pulmonary metastases and the number and size of these metastases was so large that they increased the total weight of the animal lungs.

Our subsequent analysis of lung metastases from patients with OS showed negligible expression of Fas receptor and thus confirmed our animal findings.31 Retrospective analysis of OS lung metastases from patients revealed a significant correlation between Fas expression and the administration of preoperative salvage chemotherapy.31 Using our animal models with OS lung metastases, we observed that the therapeutic efficacy of several anticancer agents was accompanied by enhanced expression of Fas in OS lung metastases and that corruption of Fas signaling significantly impaired the drugs’ therapeutic efficacy.30,32 These findings suggest that identification of agents that upregulate the expression of the Fas receptor in lung metastases may be important for developing novel therapeutic approaches for the treatment of OS lung metastases.

CIP4 as a Candidate Target for Osteosarcoma Progression

It seems likely that the Fas mechanism plays a role at the stage when OS tumor cells have already reached the lungs from their primary site. It is known that it is easier to treat the disease at the early stages than when it is already advanced. Similarly for cancer, the most effective treatment will be the one that prevents metastases formation. Therefore identification of the mechanisms that are involved in extravasation of OS cells from the bone into blood circulation, control tumor cell survival in the blood stream and final invasion, adhesion and survival in the pulmonary environment will lead to the development of the preventive treatment for metastases. MMP-9, SDF-1 factors mentioned above can be reviewed as one of these preventive targets. More recent findings with ezrin, a molecule that participate in cell-cell interactions and plays an important role in controlling cell shape (cytoskeleton), demonstrated that alterations in ezrin levels in OS cells can affect their metastatic potential in animal models.23 This finding indicated that cytoskeleton rearrangements are important for metastatic progression of OS. In fact, during metastasis tumor cells have to change their shape several times: first, their cell shape should be very flexible during cell extravasation between other cells, then their "flat" adherent shape should change to spherical non-adherent shape during its circulation in the blood stream and then rearrange accordingly back when they adhere at the metastatic site. One of the key molecules that control cytoskeleton rearrangement and maintain cell polarity is Cdc42. Cdc42 interacting protein 4 (CIP4) was first discovered in 1997 by Aspenström.34 Despite the fact the structure of CIP4 is well described (Fig. 2) and its general function in normal mammalian cells is determined as a cytoskeleton regulatory protein35-37 very little is known about its role in cancer cells. The existing in vitro data about CIP4 is controversial38-39 and needs extensive detailed study.

Figure 2: Downregulation of CIP4 expression in osteosarcoma...

Figure 2: CIP4 protein structure. CIP4 is a member of the F-BAR family of proteins which have been recently described to be involved in sensing and generating membrane curvature. At the N terminus, CIP4 contains an FCH (fes/fps/cip4 homology) domain and coiled-coil region that comprise the F-BAR domain. This domain interacts with the membrane. At the C-terminus, an SH3 domain exists and interacts with WASp, an activator of actin nucleation and polymerization). Protein tyrosine kinases such as Src or Lyn interact via their SH3 domain with a proline-rich motif found in CIP4. Also, CIP4 contains a region that binds only the activated form of Cdc42. Altogether, CIP4 behaves as a scaffolding protein involved in cytoskeletal reorganization.

In our preliminary studies with OS and breast cancer cells, we observed higher levels of CIP4 in metastatic tumor sublines than in the non-metastatic parental cells (unpublished data). We and other investigators have shown that downregulation of CIP4 in tumor cells significantly impaired their motility, invasion and adhesion in vitro.39 Based on that it would be logical to suggest that inhibition of CIP4 in tumor cells should also decrease their ability to form metastases in animal models and eventually in patients. In our future experiments with OS lung metastases animal models we are planning to study the function of CIP4 in OS tumorigenesis and metastasis. Our findings may have clinical application leading to the development of the preventive treatment for OS patients. For that it will become important to understand the mechanisms that regulate CIP4 expression in cells and what molecules are controlled by CIP4.

Grant Funding

The Liddy Shriver Sarcoma Initiative and the Foster Foundation co-funded this $50,000 grant in February 2008.

Plan Figure 1A: Alterations in the Fas Signaling changes metastatic behavior of osteosarcoma cells in vivo. (A) LM7 human osteosarcoma cells were stably transfected with control neo-plasmid (LLM7-neo) or with Fas-plasmid (LM7-Fas). Cells were injected intravenously into immunodeficient mice and after 10 weeks their lungs were examined for metastases. All mice injected with LM7 or LM7-neo cells developed numerous visible metastases in the lungs. In contrast, fewer mice injected with LM7-Fas cells developed visible lung metastases and the number and size of these metastases was significantly smaller then in LM7 and LM7-neo groups. (B) K7 mouse osteosarcoma cells were transfected with control neo-plasmid or with FADD-dominant-negative plasmid (FDN), which inhibited Fas signaling. Stably transfected control clones K7/neo1 and K7/neo5 and two clones K7/FDN1 and K7/FDN5 were selected and then injected intravenously into immunocompetent mice. After 4 weeks animal lungs were examined for metastases. K7 and K7/neo groups of mice had less than 10% incidence of visible lung metastases, whereas all mice in K7/FDN groups developed visible pulmonary metastases and the number and size of these metastases was so large that they increased the total weight of the animal lungs.

Plan Figure 1B: Alterations in the Fas Signaling changes metastatic behavior of osteosarcoma cells in vivo. (A) LM7 human osteosarcoma cells were stably transfected with control neo-plasmid (LLM7-neo) or with Fas-plasmid (LM7-Fas). Cells were injected intravenously into immunodeficient mice and after 10 weeks their lungs were examined for metastases. All mice injected with LM7 or LM7-neo cells developed numerous visible metastases in the lungs. In contrast, fewer mice injected with LM7-Fas cells developed visible lung metastases and the number and size of these metastases was significantly smaller then in LM7 and LM7-neo groups. (B) K7 mouse osteosarcoma cells were transfected with control neo-plasmid or with FADD-dominant-negative plasmid (FDN), which inhibited Fas signaling. Stably transfected control clones K7/neo1 and K7/neo5 and two clones K7/FDN1 and K7/FDN5 were selected and then injected intravenously into immunocompetent mice. After 4 weeks animal lungs were examined for metastases. K7 and K7/neo groups of mice had less than 10% incidence of visible lung metastases, whereas all mice in K7/FDN groups developed visible pulmonary metastases and the number and size of these metastases was so large that they increased the total weight of the animal lungs.

Plan Figure 2: CIP4 protein structure. CIP4 is a member of the F-BAR family of proteins which have been recently described to be involved in sensing and generating membrane curvature. At the N terminus, CIP4 contains an FCH (fes/fps/cip4 homology) domain and coiled-coil region that comprise the F-BAR domain. This domain interacts with the membrane. At the C-terminus, an SH3 domain exists and interacts with WASp, an activator of actin nucleation and polymerization). Protein tyrosine kinases such as Src or Lyn interact via their SH3 domain with a proline-rich motif found in CIP4. Also, CIP4 contains a region that binds only the activated form of Cdc42. Altogether, CIP4 behaves as a scaffolding protein involved in cytoskeletal reorganization.